Combination of intercalating organometallic complexes and tumor seeking biomolecules for DNA cleavage and radiotherapy

ABSTRACT

The invention relates to molecules for treatment and diagnosis of tumors and malignancies, comprising a tumor seeking biomolecule, which is coupled to an intercalating moiety, which is capable of complexing a metal, which metal is preferably a radioactive metal, to the use of these molecules and to therapeutic and diagnostic compositions containing them.

This application claims priority to provisional application No.60/121,340 filed Feb. 24, 1999.

BACKGROUND OF INVENTION

The present invention relates to new molecules for the treatment anddiagnosis of tumors. The invention furthermore relates to therapeuticalcompositions comprising one or more of these molecules and to the use ofboth in treatment and diagnosis of cancer.

The diagnosis and therapy of cancer still requires a large input fromthe pharmaceutical and chemical industry. Although a substantial effortis made to develop new treatments, there are still many tumor types forwhich no treatment exists. An additional problem is the formation ofmicrometastascs, which cannot be diagnosed or treated.

An important problem in treatment is the similarity between normal cellsand cancer cells. Treatments interfering with the growth of tumor cellswill also interfere in the growth of healthy cells. Radiotherapy as itis now known consists essentially of an arbitrary cross-fire fromoutside the cell or the cytoplasm. Because this is a rather roughtreatment surrounding cells and tissues might also be damaged leading tomore or less severe side effects.

The provision of an improved radiotherapy and diagnostic method forcancer which uses very low amounts of radionuclides and leads to adirect treatment in the malignant cell is therefore highly desirable.

It is known that the metabolism of cancer cells differs from that ofnormal cells. In addition, cancer cells appear to have an increasedmembrane permeability in comparison to normal cells due to an increasedexpression of membrane receptors. The result is that the cancer cellsare more permeable for biological vectors, like proteins and peptides.

The enhanced uptake of such biological vectors can be used in thediagnosis of tumors by binding a radionuclide to a protein, for exampleby iodination of tyrosine functions in the protein or by covalentcoupling of radioactive metal complexes. These molecules combine a tumorseeking function and a radioactive function. Although these types ofmolecules have been used for diagnosis, their use in therapy was not yetdescribed.

SUMMARY OF INVENTION

It is the object of the present invention to further improve on theabove described molecules to come to an even better tailored treatmentof malignant cells.

This object is achieved by the invention by the provision of a moleculein which three functions are combined. This molecule comprises a tumorseeking molecule, which is coupled to an intercalating moiety, which iscapable of complexing a metal, which metal is preferably a radioactivemetal. The molecule can be targeted specifically to the tumor by thetumor seeking molecule and be internalized by the cell. Theintercalating moiety will then insert into the DNA strand and inducebreaks. In addition, the radioactive metal will also lead to strandbreaking of the DNA. The advantage of the new molecules is that they arespecifically directed to the malignant cell and are taken up by thecell.

The tumor seeking molecule is preferably a biomolecule, such as apeptide or protein that is actively targeted to the tumor cell. Examplesof these biomolecules are somatostatin-, neurotensin-, bombesin-receptorbinding molecules, monoclonal antibodies, antennapedia peptide, e.g.,PENETRATIN®, and glycoproteins, and molecules binding to the GPIIb/IIIareceptors. PENETRATIN® is manufactured by Cyclacel Limited, having aplace of business at 5 Whitehall Crescent Dundee, Scotland DD1 4AR,United Kingdom. The invention is however not limited to these examplesand is more generally applicable to other tumor seeking agents as well.This category encompasses in addition compounds which are known to betransported into the nucleus or the nucleus membrane. Examples of theseare anti-sense oligonucleotides, proliferating agents, likedeoxy-uridine, and small molecules, like spermidine. The intercalatingmoiety is preferably an aromatic molecule with an intercalative bindingaffinity for double-stranded DNA. Examples of such aromatic compoundsare compounds containing i.e. acridine, porphyrin, ellipticine,phenantroline, carbazole, benzimidazole or compounds with knowncytostatic activity (antibiotics) from the class of tetracyclines(anthracyclines), such as daunorubicine, epirubicine or mixoxantrone andare functionalized with ligands able to coordinate the [M(CO)₃]⁺ moiety.Examples of such ligands are those mentioned in EP-879 606 andadditionally polyamino-polycarboxylates, phosphates and phosphonates,aliphatic or aromatic or mixed triamines and thiones.

The intercalating and tumor seeking functions are sometimes combined inexisting molecules. Examples of intercalating agents combining anintercalating moiety and a peptide are actinomycin and triostin.

The radioactive molecule can be any radioisotope. Pure γ-emittingnuclides are preferred since their accompanying low range conversionelectrons will lead to cleavage of bonds, which are close to thedecaying nucleus. The dose burden to the patient remains thus very low.

Particularly suitable combinations of the three functions are given inFIG. 1.

The invention further relates to the use of the molecules in therapy anddiagnosis and to therapeutical and diagnostic compositions comprisingone or more of these molecules.

Therapeutical compositions comprise at least a suitable amount of themolecule in a diluent or excipient. Such compositions can take the formof solutions and are administered intravenously, intraperitoneally orintrathecally. Suitable amounts to be administered depend on the way ofadministration, the radionuclide used and the indication to be treatedor diagnosed. Suitable amounts vary between 10⁻⁹ and 10⁻¹ g per kg bodyweight.

Excipients and diluents for this type of medication are well known tothe skilled person. However, the present molecules require certainconditions for stability. Preferably, the excipient or diluent should beof a hydrophilic and preferably organic nature.

For diagnostic purposes the composition consists of at least a suitableamount of the molecule in a diluent or excipient. Diagnostic methods tobe used with the composition of the invention are scintigraphy orMagnetic Resonance Imaging (MRI).

It was now found that the method for the synthesis of Tc and Recarbonyls from water described in EP-879 606 is suitable for preparationof the molecules of the invention. It is in particular possible withthis method to introduce intercalating ligands, which form very stablecomplexes (in vitro and in vivo) with the above mentioned carbonyls.EP-879 606 is incorporated herein by reference.

The ligands claimed in EP-879 606 and acridine, porphyrin, ellipticine,phenantroline, carbazole, benzimidazole do stabilize the fac-[Tc(CO)₃]⁺moiety in serum and form complexes at very low concentrations. Theseligands can be site specifically attached to the biomolecules andsubsequently be labeled with i.e. Tc-99m. Since the radionuclide is veryclose to the intercalating ligand, its low energy electron willpenetrate the DNA-strands very well and induce strandbreaking. Whenintercalating in one of the grooves, the probability to hit is very highsince the nucleus is practically surrounded by DNA.

The biomolecules derivatized according to the invention exhibit highselectivity and are internalized. As known from pure organicintercalators, the complex is going to intercalate in DNA in particularwhen the cell is dividing. In contrast with other therapeutics, a highselectivity can be achieved with this combination.

If Re-188 is applied as the radionuclide, the damages will be much moresevere than in the case of Tc-99m, but, consequently, the applied amountof radioactivity will be much lower than in case of “normal”radiotherapy. Thus, severe side effects such as bone marrow toxicitycould be avoided.

The present invention will be further illustrated in the examples thatfollow and which are solely intended to clarify the invention, but arein no way intended to be limiting to the scope thereof.

BRIEF DESCRIPTION OF DRAWINGS

In the Examples reference is made to the following figures:

FIG. 1: schematic representation of potential molecules of theinvention.

FIG. 2: example of a Tc(I) complex with this intercalating ligand and apotential biomolecule attached by direct linkage to another coordinationsite.

FIG. 3: schematic representation of the method for preparing moleculesof the invention.

FIG. 4: schematic representation of the three types of plasmidstructures.

FIG. 5: ethidium bromide stained agarose gel of the three types I, IIand III of DNA (left lane) and a molecular weight marker (right lane).

FIG. 6: ethidium bromide stained agarose gel of a plasmid preparationtreated with the compound [^(99m)Tc(P₁)(teta)(CO)₃]; The applicationsite of the sample is on the bottom of the gel. Lane 1 is the molecularweight marker; lane 2 is a reference solution containing supercoiled,relaxed (single strand break) and linearized (double strand break)plasmid, lane 3 is the experimental solution containing both plasmid andthe intercalator of the invention, and lane 4 is the negative referencecontaining only plasmid.

FIG. 7: reaction scheme for the preparation of model bifunctionalintercalators.

FIG. 8: reaction scheme for the preparation of model trifunctionalintercalators.

DETAILED DESCRIPTION EXAMPLES Example 1

Synthesis of the Molecules of the Invention

1. Introduction

To provide a strong intercalation, the intercalator should be preferablyplanar and aromatic heterocyclic. Furthermore, pendant groups in theintercalator must stably be coordinated to the radionuclide (i.e.^(99m)Tc). In this example, it is not coercive that the coordinatingunit must be a multidentate ligand with high thermodynamic stability,since most complexes with Tc(I) show an extremely high kineticstability. For these reasons and due to the already known principles ofcomplexation of several mono- and bidentate ligands (especiallypicolinic acid) 5,6-benzochinolin-3-carboxylic acid was selected asintercalator.

FIG. 2 depicts an example of a Tc(I) complex with this intercalatingligand and a potential biomolecule attached by direct linkage to anothercoordination site.

2. Synthesis of the Example Intercalator

2.1. 3-cyano-4-benzoyl-3,4-dihydrobenzo(f)chinoline 2

648 μl (5.58 mmol) benzoyl chloride was added to a two phase system ofwater/methylene chloride over a period of two hours. These two layerscontain 500 mg (2.79 mmol) of benzo(f)chinolin in the methylene chloridelayer and 545 mg (8.37 mmol) KCN in water. Stirring was continued for 6hours. The organic phase was separated and washed with water, 5%hydrochloric acid, water, 5% NaOH solution, and again with water. Afterdrying over magnesium sulfate, the solution was evaporated to dryness.

The bromide salt of this so-called Reissert-compound was recrystallizedfrom 95% ethanol to yield the analytically pure substance. Yield: 612 mg(71%).

2.2 5,6-benzochinolin-3-carbon acid (P1)

2 ml 48% hydrobromide acid were added to 287 mg (0.93 mmol) of theReissert-compound dissolved in 2 ml acetic acid. The solution wasrefluxed during 24 hours, cooled and filtered. The filtered product waswashed with diethyl ether, dried, and recrystallized from methanol toyield 169 mg (0.76 mmol) (82%) of the hydrobromide of the intercalatoras a yellow solid.

2.3 Macroscopic Synthesis of Technetium and Rhenium Complexes with P1(5,6-benzochinolin-3-carbon acid)

2.3.1 [NEt₄][ReBr(P1)(CO)₃]

A suspension of 102 mg (133 μmol)

[NEt₄][RcBr₃(CO)₃], 29.7 mg (133 μmol) P1 and 116 μl (226 mmol) oftrioctylamine were refluxed in dichloromethane until a clear solutionwas achieved. After evaporation of the solution, the complex 5 wasextracted into THF. After evaporization of THF the residue was washedwith diethyl ether to remove trioctyl ammonium bromide. Yield: 63 mg(67%) of the yellow complex.

2.3.2 [Re(P₁)(H₂O)(CO)₃]

200.0 mg (0.26 μmol) of [NEt₄]₂[ReBr₃(CO)₃] were refluxed in thepresence of 29.1 mg of the intercalator P1 during 4 hours in 1MMES-buffer solution. Then the yellow precipitation was filtered. Yield:114.2 mg (86%).

2.4 Microscopic Synthesis of [^(99m)Tc(H₂O)(P₁)(CO)₃]

The ^(99m)Tc complexes were synthesized in a two-step procedure with anormal generator eluate. In a first step the complex was synthesizedin >97% yield according to the literature (R. Alberto et al., J. Am.Chem. Soc. 120, 7987 (1998)). The solution was then neutralized withphosphate buffer in the reaction vial and a solution of thecorresponding ligand was added. The end concentration was between 10⁻⁴and 10⁻⁵. It was left standing for 30 minutes at 75° C. Theradio-chemical purification and the yield were defined throughHPLC-chromatography and it was discovered that[^(99m)Tc(HPO₄)(P₁)(CO)₃]²⁻ (compound 10) with a yield of 80-95%(dependent on the ligand concentration and the reaction time) wasformed.

2.5 Synthesis of Model Trifunctional Molecules of the Invention

This is an example how a trifunctional molecule can be built. Theprocedure is based on known synthetic approaches for the correspondingcoupling methods. The schematic procedure is given in FIG. 3.

1. Syntheses of the Bifunctional Ligands, Bearing an Intercalator and aCoordinating Functionality

2-Methlquinoline (1)

2-Methylquinoline (1) was bought from Fluka and used without furtherpurification.

Quinoline-2-carbaldehyde (2)

A mixture of 5.5 g of selenium dioxyde (49.5 mmol) in 50 ml dioxane and2 ml water was added in small portions over 10 minutes to a boilingsolution of 4.4 g (30.7 mmol) of 2-methylquinoline (1) in 20 ml dioxane.After 6 hours of boiling, the warm reaction mixture was filtered. Thefiltrate was evaporated, dissolved in dichloromethane and filteredthrough Alox. The yellow-brown solid product obtained after evaporationof the solvent was recrystallized from dichlormethane. Yield: 3.76 g(78%).

¹H-NMR (DMSO):δ, 10.12s, 8.61d, 8.22d, 8.12d, 7.99d, 7.91t, 7.79t

Compound 3a

A mixture of 500 mg of quinoline-2-carbaldehyde (2) (3.2 mmol) and 330mg of N-(2-aminoethyl)-acetamid (3.23 mmol) in 15 ml of methanol wasstirred for 2 hours at room temperature. The light brown solid productobtained was directly used for the next reaction.

Yield: ˜770 mg (˜100%).

¹H-NMR (CDCl₃):δ, 8.57s, 8.21d, 8.13d, 8.10d, 7.85d, 7.75t, 7.59t

Compound 3b

A solution of 175 mg (4.62 mmol) of NaBH₄ in 10 ml of ethanol was slowlyadded over 2 hours to a stirred solution of 500 mg (2.07 mmol) of 3a in30 ml ethanol at 0° C. This mixture was then stirred overnight at roomtemperature. The solid substance obtained after evaporation of thesolvent was triturated with a 3M Na₂CO₃ solution. The desired lightbrown product (3b) was then extracted with dichloromethane. Yield: 382mg (76%).

¹H-NMR (CDCl₃):δ, 8.15d, 8.05d, 7.81d, 7.71t, 7.54t, 7.35d, 6.84br,4.21s, 3.50q, 3.02t, 2.02s.

Compound 3c

A solution of 200 mg of 3b (0.82 mmol) in 20 ml of 2N HCl was refluxedfor 6 hours. The oil obtained after evaporation of the solvent waswashed with ethanol to give the desired light brown solid hydrochloridesalt 3c. Yield: 203 mg (90%).

¹H-NMR (D₂O):6, 8.40d, 7.95t, 7.76t, 7.59t, 7.49d, 4.57s, 3.46t, 3.34t

N-BOC-diethylentriamine (4)

A solution of 500 mg (2.29 mmol) of di-tert-butyl dicarbonate ((BOC)₂O)in 30 ml dioxan was slowly added to a solution of 1.49 ml (1.42 g)(13.74 mmol) of diethylentriamine in 80 ml of dioxan at 10° C. Themixture was then stirred for 15 hours at room temperature. The desiredproduct precipitated as an oil, which was then separated from the restof the solution, dissolved in water, filtered, and extracted withdichloromethane to finally give the desired product as a light yellowoil. Yield: 260 mg (56%).

¹H-NMR (CDCl₃): 5, 5.15br, 3.25br, 3.18t, 2.77t, 2.69t, 2.63t, 1.76br,1.41s, 1.19t

Compound 5a

A mixture of 140 mg of quinoline-2-carbaldehyde (2) (0.89 mmol) and 200mg of N-BOC-diethylentriamine (0.99 mmol) in 30 ml of methanol wasstirred for 3 hours at room temperature. The solid obtained afterevaporation of the solvent was then washed with water to obtain thedesired light brown product. Yield: 304 mg (94%).

¹H-NMR (DMSO):δ, 8.32d, 7.97t, 7.73t, 7.71d, 7.57t, 6.65t, 4.33s, 3.08t,2.97t, 2.85t, 1.28s, 1.09t

Compound 5b

A solution of 41 mg (1.08 mmol) of NaBH₄ in 10 ml of ethanol was slowlyadded over 2 hours to a stirred solution of 148 mg (0.43 mmol) 5a in 30ml of ethanol at 0° C. This mixture was then stirred overnight at roomtemperature. The solid brown oil obtained after evaporation of thesolvent was triturated with a 3M Na₂CO₃ solution. The desired lightbrown product (3b) was then extracted with dichloromethane. Yield: 136mg (92%).

¹H-NMR (DMSO):δ, 8.29d, 7.94d, 7.92d, 7.71t, 7.61d, 7.54t, 6.71t, 3.95s,2.96q, 2.59s, 1.33s, 1.22t

Compound 5c

A solution of 100 mg of 5b (0.29 mmol) in 3N HCl was refluxed for 2hours. The oil obtained after evaporation of the solvent was washed withdiethylether to give the desired light brown solid hydrochloride salt5c. Yield: 102 mg (94%).

¹H-NMR (D₂O):δ, 8.44d, 7.95t, 7.77t, 7.6t, 7.5 Id, 4.51s, 3.44s, 3.34t,3.27t

2. Synthesis of Trifunctional Model Intercalators

Trifunctional intercalators were prepared starting from 5a or 3b of part1 above. FIG. 8 gives the specific reaction scheme.

1. Alkylation of an Amine with bromo-aceticacid-ethylester

Amine I (FIG. 7; 547 mg, 2.83 mmol) and triethylene amine (0.510 ml,3.08 mmol) were stirred in methanol (10 ml). The solution was cooled to0° C., and ethyl bromoacetate II (0.313 ml, 2.83 mmol) was addeddropwise within 5 minutes. After stirring the solution at roomtemperature for 18 hours, the solvent was removed in vacuo. The residuewas dissolved in dichloromethane (50 ml) and washed three times withwater (20 ml). The water phases were washed twice with dichloromethane(50 ml). The organic phases were dried over MgSO₄, filtered, and thesolvent was removed in vacuo to give III as a yellow oil. Yield: 590 mg(2.11 mmol, 74.6%).

TLC (silica, ethanol) RF 0.4

¹H NMR (200 MHz, d₆-acetone) δ=8.44 (m, 1H, picolin), 7.65 (m, 1H,picolin), 7.45 (m, 1H, picolin), 7.21 (m, 1H, picolin), 4.12 (q, 2H,J=7.2 Hz, CH₂ ester), 3.94 (s, 2H, CH₂), 3.64 (s, 2H, CH₂), 3.32 (m, 2H,N—CH₂—CH₂—N), 2.82 (m, 2H, N—CH₂—CH₂—N), 1.84 (s, 3H, CH₃—CO), 1.21 (t,3H, J=7.2 Hz, CH₃ ester).

2. Deprotection

Amine III (576 mg, 1.94 mmol) was dissolved ethanol (4 ml) and water (8ml). NaOH 2M (2 ml) was added, and the solution was stirred at roomtemperature for 1.5 hours. Analytical HPLC exhibited a single peak,indicating that the ester group was cleaved quantitatively.

The solvent was removed in vacuo, the residue was dissolved in water (8ml), and HCl 2N (1 ml) was added to neutralize the solution. HCl 33%(1.0 ml) was added, and the reaction mixture was stirred at 90° C. for48 hours. NaHCO₃ was added to neutralize the reaction mixture, thesolvent was removed in vacuo and the residue was washed with ethanol.Removing of the solvent gave the deprotected product V as a yellow oil.Yield: 352 mmol (1.68 mmol, 68.6%).

¹H NMR (300 MHz, D₂O) δ=8.44 (m, 1H, picolin), 7.85 (m, 1H, picolin),7.45 (m, 1H, picolin), 7.39 (m, 1H, picolin), 3.78 (s, 2H, CH₂), 3.35(m, 2H, N—CH₂—CH₂—N), 3.22 (s, 2H, CH₂), 3.32), 2.79 (m, 2H,N—CH₂—CH₂—N).

Example 2

Strand Breaking with the Molecules of the Invention in a Model System

1. Introduction

1.1 The Use of Plasmids

To investigate the ability of the intercalating complexes with ^(99m)Tcto induce DNA-strandbreaks, plasmids were used as a model system.Plasmids are very suitable because electrophoretic analyses allow todifferentiate between double and single strand breaks. Additionally,large quantities of plasmids can be produced very simply by using cellbiological methods.

A plasmid is a circular double-stranded DNA molecule, which doublehelical axis can be drilled into a superhelix. This form of thesuperhelix is described as type I. This type may loose itssuperhelix-structure by a single strandbreak and is then present as arelaxed circular DNA (type II). Through a double strandbreak of bothtypes a linear from (type III) of the plasmid will be created. FIG. 4shows an example of the structure of these 3 DNA types.

Because these three DNA types have different structures, they may wellbe separated due to their size and especially their form byelectrophoresis on agarose gel. The mixture (type I-III after theexperiment) to be investigated is loaded on an agarose gel. A constantvoltage will then be applied and the negatively charged DNA-fragmentswill migrate toward the cathode. The larger the form of the fragment,the slower the migration along the gel. DNA of type I (most compact)moves fastest, type II slowest. The gel will then be put in solutionwhich contains very little ethidium bromide. The DNA fragments are madevisible by intercalation and irradiation with UV-light of 300 nmdepicting red-orange colored fluorescence (590 nm). This method is sosensitive that less than 5 ng DNA per band are detected. In thephotographic record of the gels in FIG. 5, the migration direction isfrom the top to the bottom.

1.2 Production of the Plasmids

The plasmid Bluescript KS™ with a size of 2958 base pairs has beenproduced following the standard protocol of the company QIAGEN. Usually,this plasmid exists in the superhelix form (type 1). With therestriction enzyme KpnI the linearized form of the plasmid DNA (typeIII) can be produced. A single strandbreak resulting in the relaxedcircular form of plasmid (type II), can be induced by the enzyme DNAaseI. FIG. 5, right lane, shows the electrophoresis on agarose-gel of amixture of these three types of DNA. For the electrophoresis a markerwith several sizes of DNA-pieces has been used as reference (left lane).

As demonstrated by the three bands of the right lane, the three types ofDNA were clearly separated and can be distinguished after visualization.If single or double strand breaks result from conversion electrons, itshould be easy detectable by this method.

2. Investigation of the Ability of [^(99m)Tc(P₁)(teta)(CO)₃] to InduceStrandbreaks

5 μl of a solution containing approx. 0.3 mCi/ml of[^(99m)Tc(P₁)(teta)(CO)₃] and 100 ng of type I plasmid (˜=3*10⁻⁵ M inbase pairs) were left standing over a period of 18 hours. Then aelectrophoresis of this mixture and the three references was made (FIG.6).

It is clearly visible that the plasmids in the measurement solution(lane 3) migrate slower than in the reference solution. The reason forthis observation is that a small change in the structure of the plasmidsis probably induced by intercalation of the complex into the doublestrand. This change in the tertiary structure of the plasmid did thenallow a better intercalation of the ethidium bromide, thus explainingthe stronger intensity of the band of sample solution.

Furthermore, in the comparison with the negative reference solution(lane 4), it is obvious that one or possibly two new bands appeared(arrows) in the lane of the solution treated with the ^(99m)Tc complex.The stronger of these two bands corresponds approximately to theposition of type II on the band of reference solution in lane 2,containing the three types. This means that the complex gas induced asingle strand break in the plasmid.

1. A compound comprising: (a) a biomolecule selected from somatostatin,neurotensin, bombesin-receptor binding molecules, antibodies,antennapedia peptide, and molecules binding to GPIIb/GPIIIa receptors;coupled to (b) an aromatic intercalating moiety with binding affinityfor double-stranded DNA selected from acridine, porphyrin, ellipticine,phenantroline, carbazole, benzimidazole, and tetracycline compounds withcytostatic activity; which is complexed to (c) a γ-emitting radioactivemetal selected from Tc-99m, Re-186, Re-188, and Mn, wherein saidcompound is associated with one or more pharmaceutically acceptableexcipients.
 2. A kit for the preparation of a diagnostic or therapeuticcomposition comprising: (a) a biomolecule molecule selected fromsomatostatin, neurotensin, bombesin-receptor binding molecules,antibodies, antennapedia peptide, and molecules binding to GPIIb/IIIareceptors; coupled to (b) an aromatic intercalating moiety with bindingaffinity for double-stranded DNA selected from acridine, porphyrin,ellipticine, phenantroline, carbazole, benzimidazole, and tetracyclinecompounds with cytostatic activity; (c) instructions to combine theabove composition with a γ-emitting radioactive metal selected fromTc-99m, Re-186, Re-188, and Mn; and (d) one or more pharmaceuticallyacceptable excipients.